The method of obtaining funds for its choleretic and anti-inflammatory activity

 

(57) Abstract:

The invention relates to a pharmacy and relates to a method of receiving funds from vegetable raw materials with choleretic and anti-inflammatory activity. Rhizomes and roots of elecampane, rhizomes and roots of Sophora yellowish, rhizomes and roots of medicinal Burnet, rhizomes of ginger, wood of Siberian elder, hawthorn blood-red and the fruit of the Apple tree berry is extracted with 25-30% ethyl alcohol in the ratio of raw materials-extractant equal to 1:(18-20) at 60-65°C. and constant stirring. The process was repeated three times. The extraction time at the 1st contact of the phases is 120 min, when the 2nd and 3rd contacts phase - 60 minutes of Extraction filter, combine, evaporated and purified by separation. The purified extract darpariaeth, dried in a vacuum dryer and milled. Yield is 22,20,6% by weight of vegetable raw materials. The invention improves the activity funds. 12 table.

The invention relates to the field of pharmacy and relates to a method of receiving funds from vegetable raw materials with choleretic and anti-inflammatory activity.

Liver disease - viral, hronicheskimi of the most dangerous and widespread. Around the world they occupy a significant place and are among the main causes of disability and mortality. In the last decade, according to the who, there is a tendency to increase the incidence.

Currently, pharmacologic treatment and prevention of diseases of the hepatobiliary system, various groups of drugs (usually synthetic), used mainly as symptomatic and, in addition, often with significant hepatotoxicity in the process of chemical transformation in the body. All this leads to the search of new drugs for the treatment of liver diseases among natural raw materials, in particular of vegetable origin, as virtually no side effects.

A method of obtaining broth from the collection "7 precious", used in Tibetan medicine as an anti-inflammatory and cleanser. For preparation of decoction collection pour water in the ratio 1:10, insist on a boiling water bath with frequent stirring for 30 min, cooled at room temperature for 10 min, filtered (pressing plant material) and add water to truboproizvoditeljam activity resources compare tool, obtained by the present method.

The objective of the invention is to increase choleretic and anti-inflammatory activity at the expense of a higher content of active ingredients (total phenolic compounds).

To achieve this goal the plant material (raw material) - rhizome and roots of elecampane (2 parts), rhizomes and roots of Sophora yellowish (2 parts), rhizomes and roots of medicinal Burnet (2 parts), rhizomes of ginger (1 part), wood elder Siberian (4 parts), the fruits of hawthorn blood-red (2 parts) and fruits of Apple and berry (part 2) - extracted with 25-30% ethyl alcohol in the ratio of raw materials-extractant equal to 1: (18-20) (adjusted with coefficient of swelling), at a temperature of 60-65oC and constant stirring. The process was repeated three times. The extraction time at the 1st contact of the phases is 120 min, when the 2nd and 3rd contacts phase - 60 minutes the Extract is filtered through serosianno cloth, unite, evaporated to 1/3 of the initial volume and purified by separation. The purified extract darpariaeth to 1/5 of the initial volume, is dried in a vacuum dryer and milled. Yield is 22,20,6% by weight of vegetable raw materials.

Pre is the first color with a pleasant odor and a bitter taste, moisture content of not more than 5%.

The method is illustrated by the following examples.

Example 1

100 g of the rhizomes and roots of elecampane, 100 g of the rhizomes and roots of Sophora yellowish, 50 g rhizomes of ginger, 200 g of wood elder Siberian and 100 g of the fruit of the hawthorn blood-red ground in a mill to a particle size diameter of 1 mm (sieve No. 10 GOST 214-83), 100 g of the rhizomes and roots of medicinal Burnet and 200 g of Apple fruit berry is crushed to particle size of 2 mm (sieve No. 20 GOST 214 - 83). The crushed raw material is loaded into the extraction apparatus with stirrer and external steam heating. Pour 13,5 l 25% ethyl alcohol in the ratio of raw material to the extractant is 1: 18 with regard to the coefficient of swelling materials. Extracted at a temperature of 60oC and continuous stirring for 120 minutes, the Extract filtered through serosianno cloth in the collection. Spend two extraction for 60 and 60 min, feeding every once in extractor 25% alcohol in a quantity equal to the amount drained retrieval. 1st drain - 10,47 L., 2nd plum - 10,50 l, 3rd plum - 10,52 L. combined extract evaporated to 1/3 of the initial volume and purified by separation. The purified extract darpariaeth to 1/5 pervonachalnogo type. Get to 163 g of the finished product, which is 21.7% by weight of the feedstock. The dry extract is a fine brown powder with a pleasant smell and a bitter taste; crumpled, the moisture content of 4.05%.

Investigated the acute toxicity of the obtained dry extract "7 precious" according to the generally accepted method of Cerberus [10]. The experiments were conducted at 35 outbred white mice of both sexes with a mass of 20-25, the Tested extract was injected once intraperitoneally at doses of 1000-5000 mg/kg of animal Observations were conducted during 14 days. Studies have shown that LD50the tested extract intraperitoneal injection is 3470 mg/kg after intragastric administration of the extract did not report the deaths of the animals and were not observed signs of intoxication during the whole period of observation that allows us to classify the test tool as a low toxic substance classification Sidorov [9].

Assessment of the choleretic activity of dry extract "7 precious" was carried out on 24 outbred white rats of both sexes with weighing 160-200 g according to the standard technique [8]. Rats were narcoticyou by intraperitoneal injection of 1% solution of Barbara of 0.8 ml/100 g weight of the animal. W for 5 hours. An aqueous solution of a dry extract "7 precious" was introduced into the duodenum syringe at a dose of 300 mg/kg to Rats of the control group was administered distilled water in the same volume. As the comparison drug used decoction collection "7 precious" made in the ratio of 1:10 [2]. Comparator drug was administered in a volume of 10 ml/kg On the extent choleretic activity was assessed by the rate of secretion and the total number of selected bile content in the bile of bile acids [4], as well as the number of allocated with bile bilirubin and cholesterol. Total cholesterol was determined by the method of C. M. drogbas [3], and bilirubin - by the method of van den Bergh in the modification of N.P. Steed [7]. Statistical processing of the obtained results by E. C. Montevista-öhringen [5].

From the data in table 1 it follows that the dry extract "7 precious" has a strong choleretic effect. In particular, the rate of bile secretion compared with the same period of the control was significantly increased by 25-31% increase in the total number of selected bile 29%. Choleretic reaction was maintained at a high level within 4 hours of experience. Along with this extract "7 precious" stimuleren 29%. In addition, the test tool was facilitated by the secretion of bilirubin from bile, the concentration of which was increased compared with the control at 21%. This choleretic activity proposed funds was higher than the comparison drug.

Anti-inflammatory activity of the obtained means were assessed by the impact on the processes of alteration and tissue regeneration. The experiments were carried out on 18 outbred white rats of both sexes with weighing 150-160 g Playback of alteration was carried out according to the method Mancina [6] by subcutaneous injection of 0.5 ml of 9% solution of acetic acid in pre-clipped region of the back; at the same time were injected intraperitoneal dextran solution at a dose of 300 mg/kg the First injection of extract 7 precious" (in the same dose as in the previous series of experiments) was carried out for 1 hour before injection of acetic acid, and then daily for 25 days. As the comparison drug used decoction collection "7 precious" made in the ratio of 1:10 [2]. Comparator drug was administered in a volume of 10 ml/kg to Rats of the control group was administered distilled water in the same volume. On 8 and 29 days of experience evaluated the area of necrotic tissue by applying a contour of necrosis in PR is 2, it follows that foreign exchange administration of the extract "7 precious" in a dose of 300 mg/kg anti-inflammatory effect, reducing the level of destruction and stimulating tissue regeneration. In particular, on the 8 day experience the infusion of the extract "7 precious" area of necrotic tissue was decreased by 33% compared with control and rats treated with the drug compared to only 15% (the difference is not significantly). On the 29th day of the experience marked a pronounced antiulcerative action of the extract "7 precious", which effectively stimulate regenerative processes, the consequence of which was the reduction of the area of necrotic tissue by 57%, whereas in rats treated with the drug comparison, the area of necrosis was decreased by 27%. Thus, the extract 7 precious" anti-inflammatory activity exceeds the comparator drug.

Example 2.

100 g of the rhizomes and roots of elecampane, 100 g of the rhizomes and roots of Sophora yellowish, 50 g rhizomes of ginger, 200 g of wood elder Siberian and 100 g of the fruit of the hawthorn blood-red ground in a mill to a particle size diameter of 1 mm (sieve No. 10 GOST 214-83), 100 g of the rhizomes and roots of medicinal Burnet and 200 g of Apple fruit berry is crushed to what LCA and external steam heating. Pour 14,25 l 30% ethanol in the ratio of raw material to the extractant is 1: 19 taking into account the coefficient of swelling materials. Extracted at a temperature of 60oC and continuous stirring for 120 minutes, the Extract filtered through serosianno cloth in the collection. Spend two extraction for 60 and 60 min, giving each time in the extractor 30% alcohol in a quantity equal to the amount drained retrieval. 1st drain - 11,20 L., 2nd plum - of 11.15 L., 3rd plum - 11,20 L. combined extract evaporated to 1/3 of the initial volume and purified by separation. The purified extract darpariaeth to 1/5 of the initial volume, is dried in a vacuum dryer at a temperature of 60oC for 8 h and ground in a mill propeller type. Get 168 g of the finished product, which is 22.4% by weight of the feedstock. The dry extract is a fine brown powder with a pleasant smell and a bitter taste; crumpled, the moisture content to 3.92%.

Assessment of the choleretic activity of dry extract "7 precious" was carried out on 24 outbred white rats of both sexes with weighing 160-200 g according to the method described in example 1.

From the table 3 data show that dry extract "7 precious" Okas what motor control, was significantly increased by 28-33% increase in the total number of selected bile 31%. Choleretic reaction was maintained at a high level within 4 hours of experience. Along with this extract "7 precious" stimulated the synthesis and secretion of bile acids, total concentration which exceeded the control by 31%. In addition, the test tool was facilitated by the secretion of bilirubin in the bile. This choleretic activity proposed funds was higher than the comparison drug.

Anti-inflammatory activity of the obtained means were assessed by the impact on the processes of alteration and tissue regeneration. The experiments were held on 18 outbred white rats of both sexes with weighing 150-160 g according to the scheme described in example 1. The results obtained are presented in table 4.

From the data presented in table 4, it follows that foreign exchange administration of the extract "7 precious" in a dose of 300 mg/kg anti-inflammatory effect, reducing the level of destruction and stimulating tissue regeneration. In particular, on the 8 day experience the infusion of the extract "7 precious" area of necrotic tissue was decreased by 34% compared with control and rats treated with the drug compared literalizing", which effectively stimulate regenerative processes, the consequence of which was the reduction of the area of necrotic tissue by 38%, whereas in rats treated with the drug comparison, the area of necrosis was decreased by 27%. Thus, the extract 7 precious" anti-inflammatory activity exceeds the comparator drug.

Example 3.

100 g of the rhizomes and roots of elecampane, 100 g of the rhizomes and roots of Sophora yellowish, 50 g rhizomes of ginger, 200 g of wood elder Siberian and 100 g of the fruit of the hawthorn blood-red ground in a mill to a particle size diameter of 1 mm (sieve No. 10 GOST 214-83), 100 g of the rhizomes and roots of medicinal Burnet and 200 g of Apple fruit berry is crushed to particle size of 2 mm (sieve No. 20 GOST 214 - 83). The crushed raw material is loaded into the extraction apparatus with stirrer and external steam heating. Pour 15,0 l 30% ethanol in the ratio of raw material to the extractant is 1: 20 taking into account the coefficient of swelling materials. Extracted at a temperature of 60oC and continuous stirring for 120 minutes, the Extract filtered through serosianno cloth in the collection. Spend two extraction for 60 and 60 min, giving each time in the extractor 30% with the s extract evaporated to 1/3 of the initial volume and purified by separation. The purified extract darpariaeth to 1/5 of the initial volume, is dried in a vacuum dryer at a temperature of 60oC for 8 h and ground in a mill propeller type. Obtain 170 g of the finished product, which is 22.7% by weight of the feedstock. The dry extract is a fine brown powder with a pleasant smell and a bitter taste; crumpled, moisture content 4,47%.

Assessment of the choleretic activity of dry extract "7 precious" was carried out on 24 outbred white rats of both sexes with weighing 160-200 g according to the method described in example 1.

From the table 5 data suggest that dry extract "7 precious" has a strong choleretic effect. In particular, the rate of bile secretion, compared with the same period of the control, was significantly increased by 29-34% increase in the total number of selected bile 31%. Choleretic reaction was maintained at a high level within 4 hours of experience. Along with this extract "7 precious" stimulated the synthesis and secretion of bile acids, total concentration which exceeded the control by 37%. In addition, the test tool was facilitated by the secretion of bilirubin in the bile. Ivanopulo activity funds received were assessed by the impact on the processes of alteration and tissue regeneration. The experiments were carried out on 18 outbred white rats of both sexes with weighing 150-160 g according to the scheme described in example 1. The results obtained are presented in table 6.

From the data presented in table 6, it follows that foreign exchange administration of the extract "7 precious" in a dose of 300 mg/kg anti-inflammatory effect, reducing the level of destruction and stimulating tissue regeneration. In particular, on the 8 day experience the infusion of the extract "7 precious" area of necrotic tissue was decreased by 31% compared with control and rats treated with the drug compared to only 15% (the difference is not significantly). On the 29th day of the experience marked a pronounced antiulcerative action of the extract "7 precious", which effectively stimulate regenerative processes, resulting in reduction of the area of necrotic tissue by 53%, whereas in rats treated with the drug comparison, the area of necrosis was decreased by 27%. Thus, the extract 7 precious" anti-inflammatory activity exceeds the comparator drug.

On the basis of qualitative phytochemical analysis as received dry extract revealed the presence of:

1. Flavonoids

1.1. 0.1 g of extract was dissolved in 50 ml of 30% this is yellow staining (flavonoids).

1.2. Two-dimensional ascending chromatography on paper stamps FN-16 in the solvent system:

A. 15% acetic acid;

B. n-Butanol-acetic acid-water (4:1:2).

When viewing the chromatogram in ultraviolet light before and after the manifestation of a 5% alcohol solution of aluminum chloride detected:

quercetin - Rf= 0,04 (A) 0,70 (B);

hyperoside - Rf= 0,36 (A), AND 0.61 (B);

identification was performed with standard samples.

2. Tannins

0.05 g of extract was dissolved in 5 ml of distilled water. To 2 ml of the resulting solution add 2-3 drops of solution salesonlinevuh alum, you receive black-and-green staining (tannins).

3. Phenol carbonic acids

Two-dimensional ascending chromatography on paper stamps FN-12 in the solvent system:

A. n-Butanol-acetic acid-water (4:1:2);

B. 15% acetic acid.

When viewing the chromatogram in ultraviolet light after spraying with ammonia vapour identified with standard samples:

coffee acid - Rf= 0,29 (A) 0,55 (B);

chlorogenic acid - Rf= 0,73 (A) 0,63 (B);

cinnamic acid - Rf= 0,64 (A), 0,32 (B).

Quantitative determination

About 0.1 g (exact naveau in a volumetric flask with a capacity of 25 ml, bring the volume of solution of 50% alcohol up to the mark. Measure the optical density of the solution in the spectrophotometer at a wavelength of 274 nm in a cell with a layer thickness of 10 mm as the reference solution using 50% ethyl alcohol. Simultaneously measure the optical density of the working standard sample (RNO) cinnamic acid. 1 ml of the prepared solution of cinnamic acid are placed in a volumetric flask with a capacity of 25 ml and the volume was adjusted solution of 50% ethyl alcohol to the mark. The comparison solution is 50% ethyl alcohol.

The content of total phenolic compounds in terms of cinnamic acid, and absolutely dry extract in percent (X) calculated by the formula:

< / BR>
where D is the optical density of the test solution;

D0- the optical density of the solution nor cinnamic acid;

m is the mass of the sample extract in grams;

m0- the mass of a sample RDF cinnamic acid in grams;

W is the loss in weight on drying of the extract in percent.

Note. Preparation of the solution nor cinnamic acid: about 0.01 g (accurately weighed) RNO cinnamic acid, previously dried at 100 ° - 105oC for 3 h, dissolved in 85 ml of 96% ethanol in a volumetric flask of 100 ml capacity is the volume of solution in the same spirit to the mark and mix.

Found: 2,450,09%.

The proposed method of producing quite simple, does not require complicated purification, allows to obtain a product of constant composition. The technology can be implemented at the enterprises producing drugs.

To identify the optimal technological regimes we have studied the main factors influencing the process of extraction of plant materials: the nature of the extractant, the ratio of raw materials, temperature, degree of crushing of raw materials, duration, and number of extraction.

Quantitative evaluation was carried out according to the output of extractive substances [1] and total phenolic compounds. Metrological characteristics are presented in table 7.

As extractants used were water and ethanol of different concentrations(10, 20, 30, 40, 50, 60, 70, 80, 96%).

As can be seen from table 8, 25-30% alcohol is the best extractant, as it allows to achieve the maximum output amount of flavonoids with regard to output extractives.

Identifying the optimal degree of grinding of raw materials was performed separately for each commodity. Quantitative evaluation was performed on the output of extractives. On La leads to contamination of the extract trudnodostupnymi high-molecular compounds, grinding each commodity should be conducted separately, to the particle size: 1 mm for the rhizomes and roots of elecampane, rhizomes and roots of Sophora yellowish, rhizomes of ginger, wood of Siberian elder, hawthorn fruit, blood-red and 2 mm for the rhizomes and roots of medicinal Burnet and Apple fruit berry.

Due to the fact that in the original plant material containing thermolabile substances (vitamins, anthocyanins, alkaloids), study of the effect of temperature on the extraction process was carried out in the temperature range from 20 to 60oC, the Data presented in table 10, the optimum temperature - 605oC.

When studying the influence of the ratio of raw materials-extractant to the extraction set (table 11) that with increasing amounts of extractant increases the yield of extractive substances and amounts of phenolic compounds, but the growth of this for ratios of 1:15 and up small, so as to prevent additional flow of extractant selected ratio equal to 1:(15-17), or, taking into account the coefficient of swelling, 1:(18-20).

To determine the duration and the required number of extraction time is set to achieve equilibrium concentrations of E. For this purpose, carried out the extraction of the crushed material 30% ethyl alcohol in a ratio of 1:18 in a water bath at a temperature of 60oC and constant stirring in a flask with reflux condenser. The extract obtained after a certain period of time(15, 30, 45, 60, 75, 90, 105, 120, 135, 150 min) during the three extraction, analyzed for the content of extractives and total phenolic compounds. It is established that the equilibrium concentration is established when the 1st contact of the phases after 120 min, with the 2nd and 3rd contacts phases after 60 min (table 12). Triple extraction provides a fairly complete depletion of raw material extracted to 86% of active substances.

The proposed method, in comparison with the known, allows to obtain the product of constant composition with a higher choleretic and anti-inflammatory activity due to the higher content of active substances (phenolic compounds).

To calculate the economic efficiency of the proposed method is currently not possible, however, the foregoing advantages, in combination with a simple scheme to obtain promote the rational use of medicinal plants and explain the perspectives of implementation of this method in the pharmaceutical industry.

Sources of information

1. The state is. the state Pharmacopoeia of the USSR: Vol. 2. General methods of analysis. Medicinal plant raw materials /the USSR Ministry of health. -11th ed., extra - M.: Medicine, 1989. - 400 C. - prototype

3. Drogbas S. M. violation of the intensity of bile production and chemical composition of bile in liver dystrophy, caused by carbon tetrachloride. /Questions of medical chemistry. - 1971. - Vol. 4. - S. 397-400.

4. Karbach J. I. Quantitative determination of bile acids in bile and blood using a chromatographic method. /Biochemistry. - 1961.-So 21, N2. -S. 305-309.

5. Montevista-öhringen E. C. Simplified mathematical-statistical methods in medical research /Pathological physiology and experimental therapy. - 1964. -N4.-S. 71 - 78.

6. Oivin I. A., SATEL S. L. a Methodology for the study of local violations of capillary permeability /materials for the pathogenesis of inflammation and pathology of blood proteins. - Dushanbe, 1961. , 49, N 5.-C. 167-173.

7. The Roller N. P. Neurohumoral mechanism choleretic action of insulin /problems of endocrinology. - 1956. -N 6.-S. 75-78.

8. The Roller N. P., Oleinik, A. N. Comparative effects of atropine and metacin on the exocrine function of the liver /Pharmacology and toxicology. - 1967. , 30, N 3.-S. 334.

9. Sido the different chemical substances. - M., 1973. - S. 47-51.

10. Requirements for pre-clinical study of the General toxic action of new pharmacological substances. - M.: Medicine, 1984.

The method of obtaining funds for its choleretic and anti-inflammatory activity, consisting of the rhizomes and roots of elecampane, rhizomes and roots of Sophora yellowish, rhizomes and roots of medicinal Burnet, rhizomes of ginger, wood of Siberian elder, hawthorn fruit, blood-red, Apple fruit berry, by extraction of plant material, wherein the plant material is extracted with 25 - 30% ethyl alcohol in the ratio of plant material - extractant equal to 1 : (18 - 20), the first time - 120 min, and the second and third - 60 min at 60 - 65oC.

 

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EFFECT: improved preparing method.

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